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The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009).
This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo.
In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.
The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer
(2017)
The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far.
In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters.
Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis.
This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.
Pocket-Proteine und E2F-Transkriptionsfaktoren regulieren die Expression von Zellzyklus-assoziierten Genen und spielen eine zentrale Rolle bei der Koordination der Zellteilung, Differenzierung und Apoptose. Störungen dieser Signalwege tragen zur Entstehung zahlreicher Tumorentitäten beim Menschen bei. Trotz der intensiven Untersuchung der Zellzyklusregulation sind viele Details noch unverstanden.
Der LIN-Komplex (LINC / DREAM) ist ein kürzlich entdeckter humaner Multiprotein-komplex, welcher dynamisch mit Pocket-Proteinen und E2F-Transkriptionsfaktoren interagiert. Eine essentielle Komponente des LIN-Komplexes ist das LIN9-Protein. Um die Funktion dieses Proteins bei der Zellzyklusregulation und Tumorentstehung genauer untersuchen zu können, wurde in unserer Arbeitsgruppe ein konditionelles Lin9-Knockout-Mausmodell etabliert.
Primäres Ziel der Arbeit war es, den Phänotyp embryonaler Fibroblasten (MEFs) aus diesen Mäusen zu charakterisieren. Bereits kurz nach Inaktivierung von Lin9 konnte ein stark verlangsamtes Zellwachstums beobachtet werden. In Lin9-depletierten MEFs wurden multiple mitotische Defekte detektiert, die u. a. strukturelle Auffälligkeiten des Spindelapparates, aberrante Zellkerne, Störungen der Chromosomensegregation sowie zytokinetische Defekte umfassen und in einer dramatischen Zunahme polyploider und aneuploider Zellen resultieren. Im Langzeitverlauf führen diese erheblichen Aberrationen zu einer vorzeitigen zellulären Seneszenz. Wird diese durch das Large T-Protoonkogen durchbrochen, können sich MEFs an den Verlust von Lin9 adaptieren, zeigen dann jedoch eine hochgradige genomische Instabilität und Substrat-unabhängiges Wachstum im Weichagar als Zeichen onkogener Transformation.
Im zweiten Abschnitt der vorliegenden Arbeit wurde die Genexpression in Lin9-defizienten MEFs mittels quantitativer Real Time-PCR untersucht um zu klären, ob die beschriebenen Defekte auf Veränderungen der transkriptionellen Aktivität zurück-zuführen sind. Dabei wurde eine erhebliche Reduktion der Expressionslevel mitotischer Gene nach Verlust von Lin9 beobachtet. Des Weiteren wurden zur Klärung der zu Grunde liegenden molekularen Mechanismen Chromatin-Immunpräzipitations-Experimente (ChIP) durchgeführt. Im Vergleich zu Kontrollzellen wurden dabei in Lin9-defizienten Zellen signifikante epigenetische Veränderungen bezüglich aktivierender Histon-Modifikationen an den Promotoren mitotischer Lin9-Zielgene festgestellt.
Im letzten Abschnitt der Arbeit sollten die Auswirkungen des heterozygoten Verlustes von Lin9 analysiert werden. Dabei zeigte sich, dass Lin9-haploinsuffiziente Zellen normal proliferieren, obwohl die Expression verschiedener G2/M-Gene leicht vermindert war. Es wurde jedoch eine Schwächung des mitotischen Spindelkontrollpunktes und in der Folge über mehrere Zellgenerationen eine Zunahme polyploider Zellen beobachtet. Mit Weichagar-Assays konnte gezeigt werden, dass bereits der heterozygote Verlust des Lin9-Gens zur onkogenen Transformation beiträgt.
Zusammengenommen dokumentieren diese Studien, dass LIN9 eine entscheidende Bedeutung bei der Regulation von Zellzyklus-assoziierten Genen spielt und sowohl einen essentiellen Faktor für die Zellproliferation darstellt als auch durch die Gewährleistung genomischer Stabilität tumorsuppressive Eigenschaften aufweist.
Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability.
Precise control of mitotic progression is vital for the maintenance of genomic integrity. Since the loss of genomic integrity is known to promote tumorigenesis, the identification of knew G2/M regulatory genes attracts great attention. LINC, a human multiprotein complex, is a transcriptional activator of a set of G2/M specific genes. By depleting LIN9 in MEFs, a core subunit of LINC, Gas2l3 was identified as a novel LINC target gene. The so far uncharacterized Gas2l3 gene encodes for a member of the family of growth arrest specific 2 (GAS2) proteins, which share a highly conserved putative actin binding CH and a putative microtubule binding GAS2 domain. In the present study GAS2L3 was identified as a LINC target gene also in human cells. Gene expression analysis revealed that GAS2L3 transcription, in contrast to all other GAS2 family members, is highly regulated during the cell cycle with highest expression in G2/M. The GAS2L3 protein showed a specific localization pattern during the M phase: In metaphase, GAS2L3 localized to the mitotic spindle, relocated to the spindle midzone microtubules in late anaphase and concentrated at the midbody in telophase where it persisted until the end of cytokinesis. Overexpression of a set of different GAS2L3 deletion mutants demonstrated that the localization to the mitotic microtubule network is dependent on the C-terminus, whereas the midbody localization is dependent on full length GAS2L3 protein. Additionally, exclusive overexpression of the CH domain induced the formation of actin stress fibers, suggesting that the CH domain is an actin binding domain. In contrast, the GAS2 domain was neither needed nor sufficient for microtubule binding, indicating that there must be an additional so far unknown microtubule binding domain in the C-terminus. Interestingly, immunoblot analysis also identified the C-terminus as the domain responsible for GAS2L3 protein instability, partially dependent on proteasomal degradation. Consistent with its specific localization pattern, GAS2L3 depletion by RNAi demonstrated its responsibility for proper mitosis and cytokinesis. GAS2L3 depletion in HeLa cells resulted in the accumulation of multinucleated cells, an indicator for chromosome mis-segregation during mitosis. Also the amount of cells in cytokinesis was enriched, indicating failures in completing the last step of cytokinesis, the abscission. Strikingly, treatment with microtubule poisons that lead to the activation of the spindle assembly checkpoint (SAC) indicated that the SAC was weakened in GAS2L3 depleted cells. Although the exact molecular mechanism is still unknown, fist experiments support the hypothesis that GAS2L3 might be a regulator of the SAC master kinase BUBR1. In conclusion, this study provides first evidence for GAS2L3 as a novel regulator of mitosis and cytokinesis and it might therefore be an important guardian against tumorigenesis.
Around 10.000 – 150.000 endogenous DNA damage-induced lesions occur in a human body per day and cell. Accumulation of unrepaired lesions can lead to aneuploidy and the loss of genomic integrity which in turn contributes to tumor formation. Therefore, an efficient DNA damage response has to be initiated, in the end leading to cell cycle inhibition and induction of repair. Since it is known that a recently characterized human multiprotein complex named LINC (or human dREAM) together with B-MYB is involved in the regulation of G2/M gene expression (Plk1, cyclin B1, cdc2 etc.), its function in the DNA damage response was analyzed in this study. In growing cells B-MYB is associated to the LIN core complex which consists of 5 different proteins named LIN-9, LIN-54, LIN-52, LIN-37 and RbAp48. After induction of DNA damage B-MYB leaves the complex and binding of E2F4 and p130 to LINC is induced. Importantly, the upstream pathway leading to LINC rearrangement is dependent on the activation of p53 and p21. Interestingly, p53 -/- cells solely have the potential to block in the G2 phase of the cell cycle, thereby making them vulnerable for errors during G2 arrest induction or maintenance. Here I demonstrate that LINC rearrangement is absent in p53 -/- cells and that B-MYB/LINC binding to target gene promoters is increased. This in turn leads to an increased G2/M gene expression after DNA damage induction and triggers premature cell cycle re-entry (checkpoint adaptation). Significantly, B-MYB expression is increased in p53 mutated primary breast cancer tumors and correlates with poor prognosis and reoccurrence probably due to its function in checkpoint adaptation. This study gives evidence that inhibition of B-MYB gene expression or B-MYB function in p53 mutant tumors could be a good choice for adjuvant therapy.
Das humane LIN-9 wurde zuerst als pRB-interagierendes Protein beschrieben und spielt eine Rolle als Tumorsuppressor im Kontext des pRB-Signalweges. Über die molekulare Funktion von LIN-9 ist jedoch wenig bekannt. Die Homologe von LIN-9 in D. melanogaster und in C. elegans, sind an der transkriptionellen Regulation verschiedener Genen beteiligt. Dies und die Tatsache, dass LIN-9 mit pRB in der Aktivierung differenzierungspezifischer Gene kooperiert, ließ vermuten, dass humanes LIN-9 einen bedeutenden Einfluss auf die transkriptionelle Regulation von Genen haben könnte. Primäres Ziel dieser Arbeit war daher die Identifizierung LIN-9 regulierter Gene. Dazu sollte mit Hilfe von cDNA-Microarray Analysen, das Genexpressionsprofil LIN-9 depletierter primärer humaner Fibroblasten (BJ ET Zellen) im Vergleich zu Kontrollzellen untersucht werden. Hierfür wurde zunächst ein RNAi-basierendes System etabliert, um die posttranskriptionelle Expression von LIN-9 in BJ-ET Zellen effizient zu reprimieren. Auf dem Ergebnis der cDNA-Microarray Analysen aufbauende Untersuchungen sollten Aufschluss über die molekularbiologische Funktion von LIN-9 geben. In dieser Arbeit konnte erstmals gezeigt werden, dass der Verlust von LIN-9 zu einer verminderten Expression einer Gruppe G2/M-spezifischer Gene führt, deren Produkte für den Eintritt in die Mitose benötigt werden. Bekannt war, dass ein Teil dieser Gene durch den Transkriptionsfaktor B-MYB koreguliert wird. Zudem konnten Untersuchungen in unserem Labor eine Interaktion von LIN-9 und B-MYB auf Proteinebene, sowie die Bindung beider Proteine an die Promotoren der LIN-9 regulierten G2/M-Gene nachweisen. Dies lässt vermuten, dass LIN-9 und B-MYB gemeinsam die Expression der G2/M-Gene kontrollieren. Die verminderte Expression von G2/M-Genen in LIN-9 bzw. B-MYB depletierten Zellen geht mit einer Reihe phänotypischer Veränderungen einher, wie einer deutlich verlangsamten Proliferation und einer Akkumulation der Zellen in der G2/M-Phase. Mit Hilfe eines Durchflusszytometers erstellte Zellzykluskinetiken ergaben, dass die Progression LIN-9 bzw. B-MYB depletierter Fibroblasten von der S-Phase durch die G2/M-Phase und in die nächste G1-Phase deutlich verzögert ist. Es konnte weder ein Arrest dieser Zellen in der Mitose noch eine veränderte Länge der S-Phase nach LIN-9 oder B-MYB Depletion festgestellt werden. Daher ist die verlangsamte Zellzyklusprogression nach LIN-9 bzw. B-MYB Verlust höchstwahrscheinlich auf einen Defekt in der späten G2-Phase zurückzuführen, welcher in einem verzögerten Eintritt in die Mitose resultiert. In D. melanogaster und in C. elegans sind die Homologe von LIN-9 und B-MYB zusammen, als Bestandteile hoch konservierter RB/E2F-Komplexe, an der Regulation von Genen entscheidend beteiligt. Daher liegt es nahe, dass im humanen System LIN-9 und B MYB ebenfalls Bestandteile eines ähnlichen Komplexes sind und dadurch die Aktivierung der LIN 9 abhängigen G2/M-Gene vermitteln. Die Tatsache, dass LIN-9 sowohl als Tumorsuppressor, als auch als positiver Regulator des Zellzyklus fungiert, lässt vermuten, dass LIN-9 zu einer stetig größer werdenden Gruppe von Proteinen gehört, welche in Abhängigkeit vom zellulären und genetischen Kontext sowohl tumorsuppressive als auch onkogene Funktionen besitzen.
Regulation of mitotic progression : Focus on Plk1 function and the novel Ska complex at kinetochores
(2006)
During mitosis the duplicated chromosomes have to be faithfully segregated into the nascent daughter cells in order to maintain genomic stability. This critical process is dependent on the rearrangement of the interphase microtubule (MT) network, resulting in the formation of a bipolar mitotic spindle. For proper chromosome segregation all chromosomes have to become connected to MTs emanating from opposite spindle poles. The MT attachment sites on the chromosomes are the kinetochores (KTs), which are also required to monitor the integrity of KT-MT interactions via the spindle assembly checkpoint (SAC). The first part of this work concerns the action of Polo-like kinase 1 (Plk1). Plk1 is one of the most prominent mitotic kinases and is involved in the regulation of multiple essential steps during mitosis consistent with its dynamic localisation to spindle poles, KTs and the central spindle. Despite a nice model of Plk1 targeting to different mitotic structures via its phosphopeptide binding Polo-box domain (PBD), the exact molecular details of Plk1 functioning, in particular at the KTs, remain obscure. By two different approaches we obtained cells with an unlocalised Plk1 kinase activity: first by generating stable HeLa S3 cell lines, which upon induction expressed the PBD and thus displaced endogenous Plk1 from its sites of action. Secondly, by rescuing cells RNAi-depleted of Plk1 with the catalytic Plk1 domain only. Centrosome maturation, bipolar spindle assembly and loss of cohesion between the chromatid arms proceeded normally in either cells, in contrast to Plk1-depleted cells, arguing that PBD-mediated targeting of Plk1 is less critical for the tested functions. Remarkably, however, both the PBD expressing as well as the Plk1-depleted cells rescued with the catalytic domain of Plk1 arrested in early mitosis in a SAC-dependent manner with uncongressed chromosomes. These data disclose a so far unrecognised role of Plk1 in proper chromosome congression and point at a particular requirement for PBD-mediated localised Plk1 activity at the KTs. In the second part of the thesis, we characterised a novel spindle and KT associated protein, termed Ska1, which was originally identified in a spindle inventory. Ska1 associated with KTs following MT attachment during prometaphase and formed a complex with at least another novel protein of identical localisation, called Ska2. Ska1 was required for Ska2 stability in vivo and depletion of either Ska1 or Ska2 resulted in the loss of both proteins from the KTs. The absence of Ska proteins did not disrupt overall KT structure but most strikingly induced cells to undergo a prolonged SAC-dependent delay in a metaphase-like state. The delay was characterised by weakened kinetochore-fibre stability, recruitment of Mad2 protein to a few KTs and the occasional loss of individual chromosomes from the metaphase plate. These data indicate that the Ska1/2 complex plays a critical role in the maintenance of a KT-MT attachments and/or SAC silencing.
Unique functions of DNA topoisomerase IIalpha and IIbeta have been suggested. A human cell line which carries a homozygeous mutation of the nuclear localization sequence of the topoisomerase IIalpha gene expresses the isoform outside the nucleus at the onset of mitosis. At mitosis topoisomerase IIbeta diffused away from the chromatin despite the nuclear lack of the IIalpha-form. Chromosome condensation and disjunction was performed with the aid of cytosolic topoisomerase IIalpha which bound to the mitotic chromatin with low affinity. Consequently an increased rate of nondisjunction is observed in these cells. It is concluded that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and that topoisomerase IIbeta does not adopt these functions. A centrosomal protein was recognized by topoisomerase IIalpha. This topoisomerase IIalpha-like protein resembles a modified form of topoisomerase IIalpha with an apparent size of 205 kDa compared to 170 kDa. The expression of the protein is constant in all stages of the cell cycle and it appears in proliferating as well as in resting cells. If there is not sufficient topoisomerase IIalpha present at mitosis the centrosomal proteins might adopt the function and a mitotic catastrophe in the cells could therefore be prevented.